This proposal presents a five-year research career development program focused on the study of the mechanism of resistance of Enterococcus faecium to the lipopeptide antibiotic daptomycin. E. faecium is known as one of the no-ESKAPE pathogens and has become one of the most challenging organisms to treat successfully in the clinical setting due to its multi-drug resistant nature, increasing prevalence, high attributable costs and limited antibiotics with reliable activity against this organism. Daptomycin is a lipopeptide antibiotic which has become the last-resort drug for the treatment of enterococcal infections due to is potent in vitro activity. However, with increasing use, the emergence of daptomycin-resistant Enterococcus is on the rise. Previous whole genome sequence comparison of a clinical strain-pair of E. faecium identified mutation in the histidine kinase of a two-component regulatory system designated YycFG, which is an essential two-component regulatory system involved in the cell envelope response to antibiotics. The role of YycFG in the development of daptomycin resistance in enterococci has not been characterized. The current proposal aims to uncover the role of the YycFG regulon in development of daptomycin resistance and design new pharmacological strategies to optimize the use of daptomycin in E. faecium isolates with alterations in the YycFG system. The following specific aims are outlined in this proposal: Specific Aim I: Characterization of the YycFG cell envelope regulon in E. faecium and Specific Aim II: Evaluation of pharmacologic strategies to treat daptomycin-resistant E. faecium. Successful completion of this work will provide insights into the genetic dynamics of resistance and cell-wall homeostasis of E. faecium and is likely to provide novel targets that could be explored therapeutically to overcome the rise of daptomycin resistance with the ultimate goal of helping clinicians improve treatment of patients infected with multidrug-resistant bacteria. Additionally, this project will serve as a platform to support the transition of a young clinician-researcher to independence whose expertise will combine bacterial molecular genetics and antimicrobial pharmacology.